Inflammatory bowel disease (IBD) results from a complex interplay of genetic, immunologic and microbial factors and is comprised of Crohn's disease and ulcerative colitis subtypes. The over 140 loci associated to Crohn's disease implicate key roles for innate immunity and macrophage regulation. Macrophages serve as critical sentinels of the immune system embedded in each tissue, providing a key switch between tolerance and activation. The immune cells that infiltrate Crohn's disease gut tissue are heavily influenced by the interplay between cytokines and key transcription factors; however, understanding of macrophage gut-based phenotype and regulatory state is presently limited. In Aim 1, we propose defining genotype-independent mechanisms modulating intestinal macrophage phenotypes. We will expand understanding of intestinal macrophage function in Crohn's disease through high dimensional mass cytometry (CyTOF) and through epigenetic analyses of human macrophages from non-inflamed and inflamed intestine. CyTOF profiling of tissue macrophages will elucidate macrophage subtypes. We have published the tissue-specific enhancer landscape in mice and propose similar studies in human intestine. We hypothesize that Crohn's disease associations will be particularly enriched within intestinal macrophage-specific enhancers and that mapping these precise correlations with altered gene expression will provide critical insights into mechanisms of disease pathogenesis. In Aim 2, we will develop of predictive network models that fully leverage naturally-occurring genetic polymorphisms (SNPs as pertubagens) to elucidate the key drivers and biological mechanisms of disease. In Aim 3, we propose defining mechanisms of macrophage phenotype and function by exploring transcription factor and autocrine cytokine pathways associated to Crohn's disease and/or identified to be regulated in studies from Aims 1 and 2. We have designed CRISPR vectors targeted to each of the 34 DNA regulatory IBD loci genes expressed in intestinal macrophages to determine their effects on macrophage hierarchies and inflammatory responses. Finally, we propose studies to define the role of IBD risk variants in macrophage responses to microbial stimuli with a particular focus on rapid post-translational proteolytic events affecting the autocrine TNF/TNFSF15 and IL1/IL18 cytokine pathways. Our multidisciplinary group with clinical, human genetic, epigenetic, computational, immunological and technology development expertise will advance understanding of the role of macrophages in Crohn's disease in a way that would not be possible by any single group alone.